Repurposing crizotinib to target RIPK1-dependent cell death.

Receptor-interacting protein kinase 1 (RIPK1) has emerged as a key regulator of cell death and inflammation, which are implicated in the pathogenesis of many inflammatory and degenerative diseases. RIPK1 is therefore a putative therapeutic target in many of these diseases. However, no pharmacological inhibitor of RIPK1-mediated cell death is currently in clinical use. Recognizing that a repurposed drug has an expedited clinical development pipeline, here we performed a high-throughput drug screen of Food and Drug Administration (FDA)-approved compounds and identified a novel use for crizotinib as an inhibitor of RIPK1-dependent cell death. Furthermore, crizotinib rescued TNF-α-induced death in mice with systemic inflammatory response syndrome. RIPK1 kinase activity was directly inhibited by crizotinib. These findings identify a new use for an established compound and are expected to accelerate drug development for RIPK1-spectrum disorders.

Quizartinib inhibits necroptosis by targeting receptor-interacting serine/threonine protein kinase 1.

Systemic inflammatory response syndrome (SIRS), at least in part driven by necroptosis, is characterized by life-threatening multiple organ failure. Blocking the progression of SIRS and consequent multiple organ dysfunction is challenging. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important cell death and inflammatory mediator, making it a potential treatment target in several diseases. Here, using a drug repurposing approach, we show that inhibiting RIPK1 is also an effective treatment for SIRS. We performed cell-based high-throughput drug screening of an US Food and Drug Administration (FDA)-approved drug library that contains 1953 drugs to identify effective inhibitors of necroptotic cell death by SYTOX green staining. Dose-response validation of the top candidate, quizartinib, was conducted in two cell lines of HT-22 and MEFs. The effect of quizartinib on necroptosis-related proteins was evaluated using western blotting, immunoprecipitation, and an in vitro RIPK1 kinase assay. The in vivo effects of quizartinib were assessed in a murine tumor necrosis factor α (TNFα)-induced SIRS model. High-throughput screening identified quizartinib as the top "hit" in the compound library that rescued cells from necroptosis in vitro. Quizartinib inhibited necroptosis by directly inhibiting RIPK1 kinase activity and blocking downstream complex IIb formation. Furthermore, quizartinib protected mice against TNFα-induced SIRS. Quizartinib, as an FDA-approved drug with proven safety and efficacy, was repurposed for targeted inhibition of RIPK1. This work provides essential preclinical data for transferring quizartinib to the treatment of RIPK1-dependent necroptosis-induced inflammatory diseases, including SIRS.

ST-segment elevations with bradycardia in inferior leads: Acute pulmonary embolism.

Electrocardiographic (ECG) abnormalities are seen in 70%-80% of patients with acute pulmonary embolism (PE). Rarely, acute PE presents with ST-segment elevation (STE) in leads II; III and aVF and V1-3 mimicking ST-segment elevation myocardial infarction (STEMI). Herein, we describe a case of acute PE presenting with STE in II; III and aVF and V1-3.

Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

OPTN variants in ALS cases: a case report of a novel mutation and literature review.

INTRODUCTION: Optineurin (OPTN)-associated mutations have been implicated in the development of type 12 amyotrophic lateral sclerosis (ALS12). We reported a case of ALS with a new OPTN variant (p.D527fs) and reviewed relevant literature to better understand the phenotypes and pathophysiological mechanisms of ALS12. METHODS: We report a case of a 55-year-old female patient with a new heterozygous variant of the OPTN gene. A literature search of ALS cases associated with the OPTN gene mutations was performed in PubMed with the search criteria as [("amyotrophic lateral sclerosis") OR ("motor neuron disease")] AND ("OPTN"). RESULTS: The case of ALS with a new OPTN variant (p.D527fs) in our report manifested with bulbar involvement in onset and a rapidly progressive course. A literature review of 37 ALS patients with OPTN mutations included 20 males and 16 females with another patient whose gender was not described. The mean onset age of 37 ALS12 patients was 48 with the youngest 23 and the oldest 83 years old. Differences in onset age between male and female patients were not significant. Mean time from initiation to death was 61.8 ± 12.0 months. Patients present with either limb onset (73.5% cases) or bulbar onset (23.5% cases). CONCLUSION: Through the literature review, we summarized the clinical characteristics of ALS12. The phenotypes of the reported patients elucidate the genetic profiles and clinical phenotypes of ALS12. Clinicians should pay close attention to the role of receptor-interacting kinase 1 (RIPK1)-dependent necroptosis in the pathophysiologic development of ALS12, since necroptosis inhibitors are expected as potential therapeutic agents for treating ALS12.

Discovery of 1,2,3-selenadiazole analogues as antifungal agents using a scaffold hopping approach.

With the increasing incidence of antifungal resistance, new antifungal agents having novel scaffolds hence are in an urgent need to combat infectious diseases caused by multidrug-resistant (MDR) pathogens. In this study, we reported the design, synthesis, and pharmacological evaluation of novel 1,2,3-selenadiazole analogues by scaffold hopping strategy. Preliminary results of antifungal activity demonstrated that the new class of compounds showed broad-spectrum fungistatic and fungicidal activity. Most importantly, these newly synthesized compounds can eliminate these azole-resistant fungi and inhibit the formation of C. albicans biofilm. In particular, compound S07 showed promising antifungal activity against five azole-resistant strains with MIC values ranging from 4 to 32 µg/mL. Then, further target identification and mechanistic studies indicated that representative compound S07 exert its inhibitory activity by inhibiting fungal lanosterol 14α-demethylase enzyme (CYP51). Interestingly, representative compounds showed low cytotoxicity on mammalian cell lines. In addition, the molecular docking studies elucidated the binding modes of these compounds toward CYP51. Altogether, these results suggest that compound S07 with novel skeleton is a promising CYP51 inhibitor for treatment of fungal infections.

Innate lymphoid cells exhibited IL-17-expressing phenotype in active tuberculosis disease.

BACKGROUND: Innate lymphoid cells (ILCs), as an important group of innate immunity, could respond rapidly to Mycobacterium tuberculosis (Mtb) infection. In this research, we studied the phenotypic changes of circulatory ILCs in active tuberculosis (TB) disease. METHODS: We recruited 40 patients with active Mtb infection (TB group) and 41 healthy subjects (NC group), and collected their clinical information and peripheral blood. Circulating ILCs, ILC subsets, dendritic cells (DCs), macrophages, and the production of cytokines in ILCs were tested by flow cytometry (FCM). Enzyme-linked immunosorbent assay (ELISA) was used to detect plasma IL-23. RESULTS: Compared with healthy control, total ILCs (0.73% vs. 0.42%, P = 0.0019), ILC1 (0.55% vs. 0.31%, P = 0.0024) and CD117+ ILC2 (0.02% vs. 0.01%, P = 0.0267) were upregulated in TB group. The total IL-17+ lymphocytes were elevated (3.83% vs. 1.76%, P = 0.0006) while the IL-22+ lymphocytes remained unchanged. Within ILC subsets, ILC3, CD117+ ILC2 and ILC1 in TB group all expressed increased IL-17 (15.15% vs. 4.55%, 19.01% vs. 4.57%, 8.79% vs. 3.87%, P < 0.0001) but similar IL-22 comparing with healthy control. TB group had more plasma IL-23 than NC group (7.551 vs. 5.564 pg/mL, P = 0.0557). Plasma IL-23 in TB group was positively correlated to IL-17+ ILC3 (r = 0.4435, P = 0.0141), IL-17+CD117+ ILC2 (r = 0.5385, P = 0.0021) and IL-17+ ILC1(r = 0.3719, P = 0.0430). TB group also had elevated DCs (9.35% vs. 6.49%, P < 0.0001) while macrophages remained unchanged. Within TB group, higher proportion of IL-17+ ILCs was related to severer inflammatory status and poorer clinical condition. CONCLUSIONS: In active TB disease, circulatory ILCs were upregulated and exhibited IL-17-expressing phenotype. This may expand the understanding of immune reaction to Mtb infection.

[Meta-analysis and systematic review of efficacy and safety of Lianhua Qingwen in adjuvant treatment of adult pneumonia].

To systematically evaluate the clinical efficacy and safety of Lianhua Qingwen in the treatment of adult pneumonia. The randomized controlled trial of Lianhua Qingwen combined with conventional Western medicine in the treatment of pneumonia were retrieved from PubMed, EMbase, Wanfang database, VIP database, and CNKI from the establishment of database to March 2020. Two researchers independently conducted literature screening and data extraction, and the third researcher was in charge of arbitration in case of any disagreement. Outcome indicators included total clinical effective rate, symptom improvement time, and incidence of adverse events. R 3.6.1 was used for Meta-analysis, and RevMan 5.3 was used for quality evaluation. Twenty-two studies were included, with a total of 2 007 patients, including 1 017 patients in the experimental group and 990 patients in the control group. The results showed that the total clinical effective rate of the experimental group was higher than that of the control group(RR=1.11, 95%CI[1.08, 1.15], P<0.001), and the antifebrile time(MD=-1.81, 95%CI[-2.42,-1.21], P<0.001), cough duration(MD=-2.32, 95%CI[-2.89,-1.76], P<0.001), rale duration(MD=-2.19, 95%CI[-2.74,-1.63], P<0.001), imaging recovery time(MD=-2.17, 95%CI[-2.76,-1.58], P<0.001) and post-treatment CRP(MD=-4.07, 95%CI[-6.39,-1.75], P<0.001] were all significantly lower than those of the control group. However, it did not proved that the experimental group was safer than the control group(RR=0.84, 95%CI[0.57, 1.24], P=0.382). The results confirmed that Lianhua Qingwen combined with conventional Western medicine in the treatment of pneumonia could improve the clinical treatment efficiency, shorten the time of fever, cough, rale disappearance and imaging recovery, improve CRP index and accelerate the recovery of pneumonia patients. However, the literatures included in this study had a low quality, and the conclusions still need to be further confirmed by more high-quality, multi-center, rigorously designed randomized controlled trial.

Molecular mechanism of azoles resistant Candida albicans in a patient with chronic mucocutaneous candidiasis.

BACKGROUND: More and more azole-resistant strains emerged through the development of acquired resistance and an epidemiological shift towards inherently less susceptible species. The mechanisms of azoles resistance of Candida albicans is very complicated. In this study, we aim to investigate the mechanism of azole-resistant C. albicans isolated from the oral cavity of a patient with chronic mucocutaneous candidiasis (CMC). CASE PRESENTATION: CMC diagnosis was given based on clinical manifestations, laboratory test findings and gene sequencing technique. Minimum inhibitory concentration (MIC) of the fungal isolate, obtained from oral cavity termed as CA-R, was obtained by in vitro anti-fungal drugs susceptibility test. To further investigate the resistant mechanisms, we verified the mutations of drug target genes (i.e. ERG11 and ERG3) by Sanger sequencing, and verified the over-expression of ERG11 and drug efflux genes (i.e. CDR1 and CDR2) by RT-PCR. A heterozygous mutation of c.1162A > G resulting in p.K388E was detected in STAT1 of the patient. The expression of CDR1 and CDR2 in CA-R was 4.28-fold and 5.25-fold higher than that of type strain SC5314, respectively. CONCLUSIONS: Up-regulation of CDR1 and CDR2 was mainly responsible for the resistance of CA-R. For CMC or other immunodeficiency patients, drug resistance monitoring is necessary.

[Application and safety evaluation of plant growth regulators in traditional Chinese medicine].

Plant growth regulator is a kind of synthetic pesticide with similar physiological activity to plant hormones. It has been widely used in grain, vegetables, fruits, flowers and other crops, and become an important technical guarantee for high yield, stable yield, high quality and efficient production of crops. In recent years, plant growth regulator is widely used in Chinese herbal medicine production for regulating the growth and development and increasing production of traditional. However the crop is different from general Chinese medicinal materials, the use of plant growth regulator should not only consider the effect of Chinese herbal medicine production, and also pay special attention to the influence of Chinese traditional medicine efficacy and safety. This paper reviewed the application of plant growth regulator in the traditional Chinese medicine, the impact on the quality and safety of Chinese medicinal materials, as well as plant growth regulator of residue limits standards and testing technology, so as to the scientific use of plant growth regulator, to promote Chinese standardization planting, provide the scientific basis to protect the safety of herbal medicine. At present, the indiscriminate use and abuse of plant growth regulators such as Zhuanggenling and bulking element are common in the production of Chinese crude drugs, which has led to a significant decline in the quality of some Chinese crude drugs, and resulted in the dual residual harm to the Chinese crude drugs and the cultivation environment, causing serious safety risks to human health. In the future, it is necessary to strengthen the registration management, use norms and limit standards of plant growth regulators in traditional Chinese medicinal materials, and strengthen the supervision and regulations on the use of fertilizer instead of medicine to avoid pesticide registration and other disorders, so as to provide a basis for the quality and safety monitoring of traditional Chinese medicinal materials. Simultaneously, it is encouraged to reduction or non-application of plant growth regulators in the production of Chinese medicinal materials, especially for traditional Chinese medicine which contains a variety of active ingredients. Therefore, it is actively advocated to cultivate Chinese medicinal materials through organic or ecological method.

Multi-residue analysis of plant growth regulators and pesticides in traditional Chinese medicines by high-performance liquid chromatography coupled with tandem mass spectrometry.

A multi-residue method was developed for the simultaneous determination of 24 plant growth regulators (PGRs) and 11 representative pesticides that were widely applied in plants used in traditional Chinese medicines (TCMs) by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The method was validated taking into consideration EU guidelines; calibration curves for all of the targeted analytes showed correlation coefficients (γ2) higher than 0.9901. The limits of detection (LOD) ranged from 0.2 to 8 µg/kg. The average recovery for all analytes in spiked samples ranged from 63.18 to 127.23%, with a relative standard deviation of ≤ 15%. The proposed method has been applied to 480 batches of TCM samples, including 34 species of medicinal plants, from the TCM market. The results showed that 14 PGRs and 5 pesticides were detected, including choline chloride, chlormequat, paclobutrazol, uniconazole, phoxim, etc. Among them, there were high detection rates for chlormequat (40%), choline chloride (100%), atonik (73.75%), abscisic acid (80.83%), and indole-3-acetic acid (41.25%). The residual level of paclobutrazol in Ophiopogonis radix exceeded the recommended maximum residue limits (MRLs) according to GB 2763-2016. In addition, 14 agrochemicals used in TCM planting were collected and detected; the result showed various PGRs were detected in samples registered as fertilizer. These results indicate that PGRs and pesticides were widely used in the cultivation of medicinal plants, especially for radix and rhizome herbs. The residue of targeted PGRs and pesticides in TCM samples from this study have a high frequency and high level. Graphical abstract.

Residue of Paclobutrazol and Its Regulatory Effects on the Secondary Metabolites of Ophiopogon japonicas.

Currently, paclobutrazol is excessively used in the planting process of Ophiopogon japonicus (O. japonicus) due to its important role in regulating the growth of tuber roots, ultimately increasing the yield and shortening the growth cycle of Ophiopogonis Radix. For insight into this process and the potential risks of paclobutrazol and its mediated consequences on the secondary metabolites in Ophiopogonis Radix, corresponding high performance liquid chromatography-tandem mass spectrometric methods (HPLC-MS/MS) were developed in this study and then applied to Ophiopogonis Radix, soil, and water samples. The results demonstrated the detection of different levels of paclobutrazol residue were in Ophiopogonis Radix, soil, and water samples. In addition, the quantitative results of the secondary metabolites showed that paclobutrazol significantly decreased four steroidal saponins in Ophiopogonis Radix, especially ophiopogonin D, where the content was decreased from 824.87 to 172.50 mg/kg. Concurrently, ophiopogonanone C, a flavonoid in Ophiopogonis Radix, also significantly decreased from 2.66 to 1.33 mg/kg. In conclusion, the residual paclobutrazol and its negative regulation on the secondary metabolism of Ophiopogonis Radix brings potential hazards to the environment and human health. These results provide more comprehensive data that can be used for the reassessment of the use of paclobutrazol in O. japonicus and the formulation of related standards.

Design, Synthesis, and Evaluation of Monoamine Oxidase A Inhibitors⁻Indocyanine Dyes Conjugates as Targeted Antitumor Agents.

Monoamine oxidase A (MAOA) is an important mitochondria-bound enzyme that catalyzes the oxidative deamination of monoamine neurotransmitters. Accumulating evidence suggests a significant association of increased MAOA expression and advanced high-grade prostate cancer (PCa) progression and metastasis. Herein, a series of novel conjugates combining the MAOA inhibitor isoniazid (INH) and tumor-targeting near-infrared (NIR) heptamethine cyanine dyes were designed and synthesized. The synthesized compounds G1⁻G13 were evaluated in vitro for their cytotoxicity against PC-3 cells using the MTT assay, and molecular docking studies were performed. Results showed that most tested compounds exhibited improved antitumor efficacy compared with INH. Moreover, conjugates G10 and G11 showed potent anticancer activity with IC50 values (0.85 and 0.4 µM respectively) comparable to that of doxorubicin (DOX). This may be attributable to the preferential accumulation of these conjugates in tumor cells. G10, G11, and G12 also demonstrated moderate MAOA inhibitory activities. This result and the results of molecular docking studies were consistent with their cytotoxicity activities. Taken together, these data suggest that a combination of the MAOA inhibitor INH with tumor-targeting heptamethine cyanine dyes may prove to be a highly promising tool for the treatment of advanced prostate cancer.

Ethanol Extract from Brucea Javanica Seed Inhibits Angiogenesis Mediated by Platelet Derived Growth Factor Receptor-beta.

The present study aimed to investigate the effects of ethanol extract from Brucea javanicaseed (EEBJS) on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and the possible molecular signal involved. Firstly, a Matrigel-based in vitro angiogenesis assay demonstrated that EEBJS inhibited the angiogenesis of HUVECs in a dose-dependent manner. Then by using porcine aortic endothelial cells which stably express human PDGFR-beta, we found that the inhibition of angiogenesis was mediated by PDGFR-beta. Taken together, we conclude that EEBJS inhibited the angiogenesis function of the vascular endothelial cells mediated by PDGFR-beta, and postulate that it might contribute to the therapeutic effects of EEBJS on malignant tumors.

Diphenyl Ethers from a Marine-Derived Aspergillus sydowii.

Six new diphenyl ethers (1⁻6) along with eleven known analogs were isolated from the ethyl acetate extract of a marine-derived Aspergillus sydowii guided by LC-UV-MS. Their structures were unambiguously characterized by HRESIMS, NMR, as well as chemical derivatization. Compounds 1 and 2 are rare diphenyl ether glycosides containing d-ribose. The absolute configuration of the sugar moieties in compounds 1⁻3 was determined by a LC-MS method. All the compounds were evaluated for their cytotoxicities against eight cancer cell lines, including 4T1, U937, PC3, HL-60, HT-29, A549, NCI-H460, and K562, and compounds 1, 5, 6, and 8⁻11 were found to exhibit selective cytotoxicity against different cancer cell lines.

Design, Synthesis and Biological Evaluation of Novel 4-Substituted Coumarin Derivatives as Antitumor Agents.

Herein, fifteen new compounds containing coumarin, 1,2,3-triazole and benzoyl- substituted arylamine moieties were designed, synthesized and tested in vitro for their anticancer activity. The results showed that all tested compounds had moderate antiproliferative activity against MDA-MB-231, a human breast cancer cell line, under both normoxic and hypoxic conditions. Furthermore, the 4-substituted coumarin linked with benzoyl 3,4-dimethoxyaniline through 1,2,3-triazole (compound 5e) displayed the most prominent antiproliferative activities with an IC50 value of 0.03 µM, about 5000 times stronger than 4-hydroxycoumarin (IC50 > 100 µM) and 20 times stronger than doxorubicin (IC50 = 0.60 µM). Meanwhile, almost all compounds revealed general enhancement of proliferation-inhibiting activity under hypoxia, contrasted with normoxia. A docking analysis showed that compound 5e had potential to inhibit carbonic anhydrase IX (CA IX).

Reprogramming Factors Remodel Melanoma Cell Phenotype by Changing Stat3 Expression.

The limited availability of melanoma stem cells is a major challenge for therapeutic reagent screening and study of molecular mechanisms. It has been shown that induced expression of four stem cell factors (Oct4, Sox2, Klf4, and c-Myc) changes the phenotype of osteosarcoma and breast cancer cells to osteosarcoma stem cells and breast cancer stem cells, respectively. The present study aimed to explore whether these four factors might change the phenotype of melanoma cells to melanoma stem cells and, if so, to examine the possible molecular signal involved. Melanoma B16-F10 cells were transfected with the plasmid TetO-FUW-OSKM which contains cDNA expressing four factors, driven by the Tet-On element. We found that expression of the four transcription factors was highly induced by DOX in the stable melanoma cell clones. Further studies confirmed that induced expression of these factors remodeled the phenotype of the melanoma cells to melanoma stem cells (MSCs). This conclusion was supported by the evidence that induced expression of these factors increased the numbers of tumor-initiating cells, (namely MSCs), both in an in vitro cell culture system and in a mouse in vivo model. The conclusion was further supported by the observation that the induction of these factors exclusively increased the mRNA of signal transducer and activator of transcription 3 which has been reported to play a crucial role in stem cell maintenance. Thus, phenotypic remodeling of melanoma cells following the induction of these four factors provided a simple and optimal means to constantly obtain MSCs for screening new therapeutic reagents. The result also reveals that Stat3 may be a crucial link between the induction of the four factors and the cell remodeling, suggesting its potential role as a target to fight melanoma.

High quality in vitro expansion of human endothelial progenitor cells of human umbilical vein origin.

The limited availability of qualified endothelial progenitor cells (EPCs) is a major challenge for regenerative medicine. In the present study, we isolated human EPCs from human umbilical vein endothelial cells (HUVECs) by using magnetic micro-beads coated with an antibody against human CD34. Flow cytometric assay showed that majority of these cells expressed VEGFR2 (KDR), CD34 and CD133, three molecular markers for early EPCs. It was also found that a bioreactor micro-carrier cell culture system (bio-MCCS) was superior to dish culture for in vitro expansion of EPCs. It expanded more EPCs which were in the early stage, as shown by the expression of characteristic molecular markers and had better angiogenic potential, as shown by matrix-gel based in vitro angiogenesis assay. These results suggest that HUVECs might be a novel promising resource of EPCs for regenerative medicine and that a bio-MCCS cell culture system might be broadly used for in vitro expansion of EPCs.

OCT4 Remodels the Phenotype and Promotes Angiogenesis of HUVECs by Changing the Gene Expression Profile.

It has been shown that forced expression of four mouse stem cell factors (OCT4, Sox2, Klf4, and c-Myc) changed the phenotype of rat endothelial cells to vascular progenitor cells. The present study aimed to explore whether the expression of OCT4 alone might change the phenotype of human umbilical vein endothelial cells (HUVECs) to endothelial progenitor cells and, if so, to examine the possible mechanism involved. A Matrigel-based in vitro angiogenesis assay was used to evaluate the angiogenesis of the cells; the gene expression profile was analyzed by an oligonucleotide probe-based gene array chip and validated by RT-QPCR. The cellular functions of the mRNAs altered by OCT4 were analyzed with Gene Ontology. We found that induced ectopic expression of mouse OCT4 in HUVECs significantly enhanced angiogenesis of the cells, broadly changed the gene expression profile and particularly increased the expression of CD133, CD34, and VEGFR2 (KDR) which are characteristic marker molecules for endothelial progenitor cells (EPCs). Furthermore by analyzing the cellular functions that were targeted by the mRNAs altered by OCT4 we found that stem cell maintenance and cell differentiation were among the top functional response targeted by up-regulated and down-regulated mRNAs upon forced expression of OCT4. These results support the argument that OCT4 remodels the phenotype of HUVECs from endothelial cells to EPCs by up-regulating the genes responsible for stem cell maintenance and down-regulating the genes for cell differentiation.

Arsenic Trioxide Activate Transcription of Heme Oxygenase-1 by Promoting Nuclear Translocation of NFE2L2.

In a previous study, we found that induced expression of Heme Oxygenase-1 (HO-1) is responsible for the resistance of human osteosarcoma MG63 cells to the chemotherapeutic agent arsenic trioxide (ATO). The present study was aimed at investigating the molecular mechanisms underlying the induction of HO-1 that occurs after exposure of MG63 cells to ATO. First, using RT-QPCT and Western-blot, we found that ATO strongly induced the expression of heme oxygenase-1 (HO-1) in these human osteosarcoma cells. Then by analyzing HO-1 mRNA of MG63 cells exposed to ATO in the presence and absence of a transcription inhibitor Actinomycin-D (Act-D), we demonstrated that ATO activates HO-1 expression in MG63 cells by regulating the transcription of the gene. Finally, through the analysis of the NFE2L2 protein levels among the total cellular and nuclear proteins by Western-blot and Immunocytochemical staning, we determined that ATO enhanced the nuclear translocation of nuclear factor erythroid 2-like 2 (NFE2L2), also known as Nrf2. From these results we have concluded that transcription activation of HO-1 resulting from the nuclear translocation of NFE2L2 is the underlying molecular mechanism for its high induction, which, in turn, is responsible for the resistance of human osteosarcoma cells to ATO treatment.